The long-term goal of this research is to determine the molecular and structural mechanisms for muscle contraction, with specific emphasis on the functional dynamics of the skeletal and cardiac myosin light chains (LC) isoforms and their role in muscle disease. Structural dynamics of the myosin LC domain will be investigated directly by using two complementary spectroscopic techniques: site-directed spinlabeling (SDSL) electron paramagnetic resonance (EPR) and fluorescence resonance energy transfer (FRET). Site-directed mutagenesis will be used to engineer specific cysteines for labeling the regulatory LC (RLC) and essential LC (ELC), which will be reconstituted into muscle fibers or purified myosin. The research is designed to provide insight in two fundamental areas of muscle research that are less well known (Aims 1 and 2) and into muscle dysfunction due to disease-causing mutations (Aims 3 and 4). The latter two aims will focus on myosin LC mutations that cause the human muscle disease, familial hypertrophic cardiomyopathy (FHC). AIM 1. Determine whether the myosin light chain (LC) domain rotates as a rigid body during muscle contraction. EPR will be used to measure angular motions and FRET to measure distance changes. AIM 2. Map the structural dynamics and interaction sites of the N-termini of RLC and ELC during muscle contraction, using SDSL EPR and FRET. AIM 3. Determine the effects of FHC-RLC mutations on the structural dynamics of the LC domain in contracting muscle using EPR. AIM 4. Determine the effects of myosin FHC-ELC mutations on the structural dynamics of myosin in muscle fibers and in solution with actin using EPR and FRET. The overall goal is to develop and test hypotheses for the role of LC structure and dynamics in muscle function and disease. A central hypothesis is that an accurate description of muscle disease phenotypes, which is needed for the effective development of therapeutic strategies, requires a quantitative characterization of protein structural dynamics and function, which this proposal will accomplish.